The phospho‐opsin Phosphatase from Bovine Rod Outer Segments

Abstract

The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin-11) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type-2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho-opsin phosphatase were purified 2100-fold and 550-fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type-2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B subunit) and 65 kDa (A subunit), both of which were of a subtype; phospho-opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type-2A protein phosphatase, i.e. PP2A,. The dephosphorylation of phospho-opsin by both fractions was found to be stimulated (4-8-fold) by the presence of protamine sulphate (250 pg/ml; 50 pM). However, when phosphopeptides corresponding to the C-terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine ; at equivalent concentrations of substrates the phospho-peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho-opsin (in the presence of protamine). It was shown that type-1 phosphatases had little activity against these phospho-peptides. Furthermore, if phospho-opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine-treated substrate with 1 M NaCI, whence the protaminedependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7 : 1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate-directed phenomenon and a hypothetical mechanism for this effect is considered.Bovine rhodopsin is composed of 11-cis-retinal linked to the protein opsin via a Schiff-base linkage [l-51 involving Lys296 [6-91. The protein structure is organised as seven transmembrane segments [7,8, 101 held together by a central disulphide bond between CysllO and Cys187 [9, 111, while its C-terminal phosp...

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

“…These results support the identification of the GRP as an oligomeric form of PP-2A and suggest a potential subunit structure of ABaC. Interestingly, PP-2A of this subunit composition, the most prevalent soluble form of PP-2A in bovine brain extracts (27), was recently identified as the principle soluble rhodopsin phosphatase of rod outer segments (28). However, we observe negligible soluble GRP activity.…”

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

“…The PP2A enzyme assay was optimized for phosducin dephosphorylation and may not have contained all the essential factors necessary for optimal opsin dephosphorylation. Protamine and dopamine have been shown to influence opsin dephosphorylation by PP2A (24,77). It has been suggested that, before Ser334 of opsin can be dephosphorylated, opsin must first be regenerated with 11-cis-retinal (78).…”

Calcium‐activated Opsin Phosphatase Activity in Retinal Rod Outer Segments

“…The stimulation of PP2A activity by trifluoperazine observed in our experiments is in contrast with the observations of Stewart et al (1983) who found no effect of trifluoperazine on PP2A activity towards protein phosphatase inhibitor-I . Substrate-related effects, similar to those described for stimulation of PP2A activity towards phosphoopsin by protamine (King et al, 1994), could account for this discrepancy. Thus, some properties of CAOP resemble those of PP2B (activation by Ca'+, sensitivity to trifluoperazine).…”